A TRANSGENE WITH REPEATED DNA CAUSES HIGH-FREQUENCY, POSTTRANSCRIPTIONAL SUPPRESSION OF ACC-OXIDASE GENE-EXPRESSION IN TOMATO

Gene silencing with sense genes is an important method for down-regula ting the expression of endogenous plant genes, but the frequency of si lencing is unpredictable. Fifteen per cent of tomato plants transforme d with a 35S-ACC-oxidase (ACO1) sense gene had reduced ACC-oxidase act ivity. However, 96% of plants transformed with an ACC-oxidase sense ge ne, containing two additional upstream inverted copies of its 5' untra nslated region, exhibited reduced ACC-oxidase activity compared to wil dtype plants. In the three plants chosen for analysis, there were subs tantially reduced amounts of both endogenous and transgenic ACO RNA, i ndicating that this was an example of co suppression. Ribonuclease pro tection assays using probes spanning intron-exon borders showed that t he reduced accumulation of endogenous ACO mRNA occurred post-transcrip tionally since the abundance of unprocessed transcripts was not affect ed. The ACO1 transgene with the repeated 5'UTR also strongly inhibited the accumulation of RNA from the related ACO2 gene in flowers, althou gh there is little homology between the 5'UTRs of ACO1 and ACO2. These results indicate that although repeated DNA in a transgene greatly en hances the probability of gene silencing of an endogenous gene, it als o involves generation of a trans-acting silencing signal produced, at least partly, from sequences external to the repeat.